CN1332713C - Methods for inhibiting brain tumor growth - Google Patents

Abstract

_The present invention describes methods for inhibiting the growth of brain tumors _{using antagonists of integrin αvβ3 or αvβ5} . The integrin antagonist in the present invention can inhibit the angiogenesis of brain tumor tissue. They also inhibited vitronectin- and tenascin-mediated adhesion and migration in brain tumor cells. They could go a step further and induce direct brain tumor cell death.

Description

Ways to inhibit the growth of brain tumors

This application is a divisional application of the application number 00805997.7 submitted on January 26, 2000.

related application

This application claims priority to Provisional Application Serial No. 60/118,126, filed February 1, 1999, which is incorporated herein by reference.

technical field

The present invention relates generally to the inhibition of tumor growth, particularly brain tumors.

Background technique

Individual references throughout this application are shown in parentheses. All of the above publications are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. A full bibliography of all of the above references is found at the end of the application, before the claims.

Like other solid tumors, brain tumors require an increasing blood supply to sustain their continued growth beyond 1-2 mm ³ (1, 2). This is achieved through angiogenesis. Angiogenesis is a process in response to endothelial growth factor released by tumor cells. The process includes: induction of endothelial cell proliferation in the quiescent microvasculature; migration of the nascent endothelium towards the tumor bed; and finally maturation to form a new capillary network (3). Brain tumors are the most angiogenic of all human neoplasms. In situ hybridization or specific antibody tests on tissue sections of patients with glioblastoma and medulloblastoma demonstrated that the main angiogenic factors are vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) ( 4-7). Glioblastoma and medulloblastoma are the most common malignant brain tumors. Indeed, VEGF expression and microvessel density in gliomas are directly related to the degree of malignant features and overall prognosis (8-9).

Emerging evidence suggests that angiogenesis is regulated by integrin activity in endothelial cells. Integrins are a family of transmembrane receptors that direct cell adhesion to extracellular matrix (ECM) proteins by binding to the amino acid sequence Arg-Gly-Asp(RGD) (10). The expression of integrins α _v β ₃ and α _v β ₅ in endothelial cells was upregulated by bFGF and VEGF, respectively (11-13). Expression of integrins _αvβ3 and _αvβ5 has _been found in glioblastoma and its associated vascular _endothelium (14, 15). Integrin-mediated adhesion results in the propagation of intracellular signals that promote cell survival, proliferation, motility, and capillary formation (16, 17). Failure of these integrins to bind their ligands will lead to apoptosis of endothelial cells (18, 19). The stromal glycoprotein vitronectin, produced by the leading invasive edge of malignant gliomas, acts as a ligand for the integrins _αvβ3 and _αvβ5 (15, ₂₀ ) _. At the same time, these findings also illustrate a complex paracrine interaction between tumor cells, brain ECM, and endothelial integrins to maintain sustained angiogenesis and growth of malignant brain tumors.

Studies using the anti- _αvβ3 antibody LM-609 or the _αvβ3 and _αvβ5 RGD cyclic peptide antagonists that block the endothelin _- ECM _interaction demonstrated that in chick chorioallantoic membranes ₍ CAM) and an anti-angiogenic response in mouse chimera models (21-23). Other factors acting on selective sites in the angiogenic pathway, such as antibodies to VEGF or their tyrosine kinase receptor ligands, are also effective in inhibiting angiogenesis (24, 25).

Existing studies have shown that the adhesion of breast cancer, melanoma and HT29-D4 colon adenocarcinoma cells to vitronectin is dependent on _αv , _αvβ3 and _{αvβ5 , respectively} (51-53). Vitronectin, produced by tumors and endothelial cells _, is recognized by _αvβ3 and _αvβ5 and is an ECM protein found at sites of tumor invasion _and neovascularization. In addition to supporting endothelial cell survival through _αv junctions and angiogenesis, vitronectin _expression can further enhance the adhesion between _tumors and endothelial cells expressing _αvβ3 and _αvβ5 integrins, thereby promoting tumor aggressiveness. In a study using _a SCID mouse/human chimera model of breast cancer, tumor aggressiveness was significantly reduced after _application of the anti- _αvβ3 antibody LM-609, suggesting that blocking _αvβ3 can directly Affect the biological characteristics of tumor cells.

Due to their high degree of invasiveness and angiogenesis, brain tumors provide an excellent model to further study the importance of integrins in tumor progression. Several studies have shown that microvessel density is correlated with the malignancy and prognosis of astrocytoma (57-59). Angiogenesis inhibitors, such as TNP-470, thrombostimulating hormone-1, and platelet factor-4, have been used in experimental brain tumors and have been shown to inhibit tumor growth (60-62). However, to date, no studies have examined the effects of integrin antagonism on brain tumor invasiveness and angiogenesis. Therefore, there is a need to study this effect and thus provide a new method of treating brain tumors.

Contents of the invention

The present invention is based on the surprising discovery that targeted antagonism against integrins, especially _αvβ3 and _{αvβ5 ,} can significantly inhibit brain tumorigenesis in vivo _. The microenvironment of brain tumors is critical to tumor behavior and determines its responsiveness to the aforementioned biologically targeted therapies. Antagonism against integrins can exert an anti-tumor effect independent of anti-angiogenic effect and may synergistically prevent tumor growth. For example, a finding of the present invention shows that antagonism against integrins can directly induce brain tumor cell death.

Accordingly, one aspect of the present invention is to provide a method of inhibiting the growth of a brain tumor in a host. The method comprises administering to a host in need of such inhibition a therapeutically effective amount of an integrin antagonist.

In one embodiment of _the invention _, the integrin may be _αvβ3 or _αvβ5 . The antagonist may be _a polypeptide antagonist of _αv , an anti _{- αvβ3} antibody, an anti- _αvβ5 antibody or a cocktail of _antibodies against _αvβ3 or _{αvβ5 respectively} .

Another aspect of the present invention is to provide a method of angiogenesis in brain tumor tissue of a host. The method comprises administering to the host a composition comprising an angiogenesis-inhibiting amount of an _αv integrin antagonist.

In one embodiment _{of the} invention, the integrin is _αvβ3 or _αvβ5 . The antagonist is _a polypeptide antagonist of _αv , an anti- _αvβ3 antibody _{, an} anti- _{αvβ5 antibody} or a cocktail of antibodies against _αvβ3 or _αvβ5 respectively.

Another aspect of the present invention is to provide a method of inhibiting ECM-dependent cell adhesion of brain tumor cells growing in the brain of a host. The method comprises administering to _the host a therapeutically effective amount of an _αv integrin antagonist _, ie, _αvβ3 or _αvβ5 integrin. In one embodiment _of the invention, the antagonist is a polypeptide antagonist of _αv or a cocktail of antibodies against _αvβ3 or _{αvβ5 ,} respectively.

Another aspect of the present invention is to provide a method of inhibiting vitronectin-dependent cell migration in brain tumor cells growing in the brain of a host. The method comprises administering to the host a therapeutically effective amount of an α _v integrin antagonist.

In one embodiment of the invention, _the antagonist is a polypeptide antagonist of _αv or an anti- _αvβ3 antibody.

Another aspect of the present invention is to provide a method of inducing apoptosis in tumor cells growing in the brain of a host. The method comprises administering to the host a therapeutically effective amount of an integrin antagonist.

In one embodiment of _the invention _, the integrin may be _αvβ3 or _αvβ5 . Antagonists may be polypeptide antagonists of _αv .

The method of the present invention is very suitable for in vivo brain tumor treatment. The present invention provides a novel therapeutic approach for the treatment of brain tumors.

Description of drawings

The above-mentioned and other features of the present invention and the method of obtaining them will become apparent and at the same time readily understood by reference to the following description and associated drawings. These figures depict typical embodiments of the invention and do not limit its scope. The specific features and details are as follows:

Figures 1a and 1b show the inhibitory effect of _αv antagonists on angiogenesis (a) and tumor growth (b) on CAMs.

Figure 2 shows tumor size (A) and mouse survival (B) after intracerebral injection of DAOY and U87MG cells.

Figures 3a and 3b show the histopathology of orthotopically injected brain tumor cells DAOY (a) and U87MG (b) treated daily with inactive (A) peptide or active peptide (BD). Huge intracerebral tumors (indicated by arrows) were seen in control animals (A), whereas no tumors or only residual tumors (indicated by arrows) were visible under the microscope in animals treated with _αv antagonists (C and D).

Figure 4 shows the survival of mice with orthotopically implanted brain tumors treated with active or inactive peptides.

Figure 5 shows the effect of _αv antagonists on orthotopically (brain) and ectopically (subcutaneous tissue) grown DAOY cells.

Figures 6a-6d show the distribution curves of integrins and the effects of _αv antagonists on the adhesion (b), migration (c) and viability (d) of brain tumor cells and brain capillary endothelial cells on vitronectin.

Figure 7 shows the effect of cyclic pentapeptide on the adhesion ability of tumor cells on ECM protein.

Figure 8 shows the effect of adhesion inhibition leading to cell death (apoptosis) in brain tumor cells and brain capillary cells. This effect was limited to ECM vitronectin and tenascin.

Figure 9 shows the results of immunohistochemistry of U87 tumor cells transplanted into the forebrain of nude mice and treated with active (anti-α _v ) or control peptide.

Detailed description of the invention

One aspect of the present invention is to provide a method for inhibiting the growth of a brain tumor in a host, comprising applying a therapeutically effective amount of an integrin antagonist to the host in need of the above-mentioned inhibitory effect.

Since the growth of brain tumors requires the interaction of integrins with their ligands, the methods of the present invention can be used to treat any tumor growing in the brain. Examples of such tumors include, but are not limited to: glioblastoma, medulloblastoma (astrocytoma, other primitive neuroectodermal and brainstem malignant gliomas). For the purposes of the present invention, it is preferred that the tumor growth is localized within the brain of the host's brain. The host can be any mammal, including but not limited to: rats and humans. If growth is impaired by treatment, tumor growth is inhibited.

For purposes of the present invention, an integrin may be any member of a specific family of heterodimeric transmembrane receptors. The receptor directs cell adhesion by binding to the amino acid sequence Arg-Gly-Asp (RGD). The receptors are expressed on both tumor cells and normal cells. The properties of integrins are well understood and clearly described in the art, as detailed in references cited (1, 2), the contents of which are hereby incorporated by reference. Examples of integrins include, but _{are not limited to: the αv family such as αvβ3 , αvβ5 , αvβ1 , αvβ6 , αvβ8 ,} and the _like .

An integrin antagonist is a molecule that blocks or inhibits the physiological and pharmacological activity of integrin by inhibiting the binding of integrin as a receptor to its ligand. The ligands of integrins are various matrix proteins, including but not limited to: vitronectin, tenascin, fibronectin and collagen I. In one embodiment of the present invention _, the antagonist _of integrin may be an antagonist of integrin _αvβ3 or integrin _αvβ5 . Preferred integrin antagonists are either monoclonal antibodies, fragments of monoclonal antibodies, or peptides.

For example, an antagonist of _αvβ3 can be any factor that inhibits the binding _{of αvβ3} to one of _its ligands, vitronectin or tenascin. Examples of _αvβ3 antagonists are described in PCT publications WO 96/37492 and WO 97/45137, the _relevant contents of which are incorporated herein by reference.

Likewise, an antagonist of _αvβ5 may be _any factor that inhibits the binding _{of αvβ5} to its ligand, vitronectin. Examples of _αvβ5 antagonists are described in PCT publication WO 97/06791, the relevant contents of _which are incorporated herein by reference.

In one embodiment of the invention, the antagonist is a polypeptide antagonist of _{αv ,} ie _a polypeptide antagonist of _αvβ3 and _αvβ5 . The polypeptide is preferably an Arg-Gly-Asp (RGD)-containing polypeptide. In one embodiment, the polypeptide antagonist is an RGD cyclic pentapeptide antagonist of _αv .

According to another embodiment of _the invention, _the antagonist may be an immunospecific monoclonal antibody to _αvβ3 or _αvβ5 . Alternatively, it may be a mixture of antibodies against _αvβ3 and _{αvβ5 ,} respectively _. In one embodiment, the _αvβ3 immunospecific monoclonal antibody has the immunoreactive characteristics of the monoclonal antibody designated LM-609 _. In another embodiment of the invention, the _αvβ5 immunospecific monoclonal antibody has the immunoreactive characteristics of the monoclonal antibody designated P1-F6 _. LM-609 and P1-F6 antibodies are well known in the industry and are commercially available from Chemicon, Temecula, CA, USA.

For the purposes of the present invention, antagonists may be used alone or in combination in the method of the present invention for inhibiting tumor growth in the brain.

Therapeutically effective dose refers to the dose of antagonist sufficient to produce measurable inhibitory effect on tumor growth in the brain of the host when the host is treated. Inhibition of tumor growth can be determined by post-staining microscopy as described herein, by MRI scans of mouse brains, or by 3H-thymidine incorporation methods familiar to those skilled in the art.

Integrin antagonists can take RGD-containing peptides, anti- _αvβ3 monoclonal _{antibodies and fragments thereof, anti-αvβ5 monoclonal antibodies and} fragments _thereof , or anti- _αvβ3 and _αvβ5 monoclonal In the case of a mixture of antibodies, etc., it should be recognized that the potency and performance of the "therapeutically effective amount" may vary. However, one skilled in the art can readily assess the potency of candidate antagonists of the invention, as demonstrated by this assay.

Antagonist potency can be measured in a variety of ways including, but not limited to, angiogenesis inhibition in the CAM assay described here, in vivo brain tumor _assays , and the detection of natural ligands and integrins such as _αvβ3 or _αv Measurement of binding inhibition between _β5 and other assays.

Dosage ranges for use with antagonists depend on the type of antagonist and its potency for the particular integrin. Suitable dosages for a particular antagonist can be found by those skilled in the art without undue experimentation in view of the present disclosure. The dose should be large enough to produce the desired effect of inhibiting tumor growth in the brain. The dose should not be too large, which may cause adverse reactions, such as cerebral edema or rapid release of cytokines in brain tumors leading to kachexia, for example, when an integrin antagonist of the present invention is used in the form of a polypeptide. The dose per kilogram of body weight ranges from 1-20 mg, applied once or several times a day, for one or several days or for a long-term application. When an integrin antagonist of the present invention is applied in the form of monoclonal antibody, the dosage range is 1-20 mg/kg, once a day to twice a week for long-term application.

The polypeptide or monoclonal antibody of the present invention is administered parenterally by injection or slow infusion over a certain period of time. Although tissues are often treated by intraperitoneal or subcutaneous administration, the antagonists of the present invention may also be administered by intraocular, intravenous, intramuscular, intracavity and transdermal routes, and may also be administered peristaltic.

The compositions are administered in a dosage formulation compatible manner and in a therapeutically effective amount. Dosage and timing of administration will depend upon the subject being treated, the subject's ability to utilize the active ingredient systemically and the extent of the desired therapeutic effect expected to be achieved. The precise dosage of active ingredient to be employed depends on the judgment of the physician and will vary from individual to individual. However, appropriate dosage ranges for systemic use will be disclosed herein, which dosage ranges will depend on the route of administration. Appropriate dosing regimens also vary, but a typical regimen is an initial dose followed by repeated doses by serial injections or otherwise at intervals of one or several hours.

According to one embodiment of the invention, the invention also provides a pharmaceutical composition useful for practicing the methods of treatment described herein. The composition comprises an antagonist of the present invention and a pharmaceutically acceptable carrier. As used herein, the phrases "pharmaceutically acceptable," "physiologically tolerable," and grammatical variations referring to compositions, carriers, diluents, and agents are all used interchangeably , used to describe that these substances can be used in mammals without causing unexpected physiological effects.

The preparations for parenteral application of the peptide or antibody of the present invention include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and organic esters for injection such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (eg, Ringer's dextrose-based solutions), and the like. There may also be preservatives and other additives, such as fungicides, antioxidants, chelating agents and inert gases.

Another aspect of the present invention is to provide a method of inhibiting angiogenesis in tumor tissue in the brain of a host. The method comprises applying to the host a composition comprising an angiogenesis-inhibiting amount of an integrin inhibitor.

As discussed in the Background section, angiogenesis, the formation of a new vascular network on top of the host's pre-existing vessels, is essential for tumor growth beyond 1–2 mm ³ . For purposes of the present invention, angiogenesis will be inhibited if angiogenesis and disease symptoms mediated by angiogenesis are ameliorated.

In one embodiment of the invention, the tumor tissue is located within the brain of the host. The host can be any mammal. Examples of hosts include, but are not limited to, mice, rats, and humans.

The dosage range for use of the antagonist will depend on the form of the antagonist and its potency for the particular integrin. Appropriate dosages for a particular antagonist can be determined by those skilled in the art without undue experimentation in view of the present disclosure. Doses should be sufficiently large to produce the desired effect of amelioration of angiogenesis and disease symptoms mediated by angiogenesis. The dose should also not be so high as to cause adverse reactions such as cerebral edema due to cytokine release due to tumor lysis or hemorrhage.

In one embodiment _of the invention _, the antagonist of integrin may be an antagonist of integrin _αvβ3 or _αvβ5 . Preferred integrin antagonists may be monoclonal antibodies or peptides. In one embodiment of the invention, the antagonist is _a polypeptide antagonist of integrin _αv , a polypeptide antagonist _of integrin _αvβ3 and _αvβ5 . Among them, the polypeptide preferably comprises Arg-Gly-Asp (RGD). In one embodiment of the invention, the polypeptide antagonist is an RGD cyclic pentapeptide antagonist of integrin alpha _v .

According to another embodiment _of the present invention, the antagonist may be a mixture of antibodies against integrin _αvβ3 and antibodies _{against αvβ3} and _αvβ5 respectively _.

Therapeutically effective dose refers to the dose of the antagonist capable of producing a detectable inhibitory effect on angiogenesis in the treated tissue, that is, the dose of angiogenesis inhibitor. Inhibition of angiogenesis can be detected in situ by immunohistochemical methods described herein or other methods familiar to those skilled in the art.

According to one embodiment of the invention, the invention also provides a pharmaceutical composition useful for the methods of treatment described herein. The composition comprises an antagonist of the present invention and a pharmaceutically acceptable carrier.

Another aspect of the present invention provides a method for inhibiting ECM-dependent cell adhesion between brain tumor cells in the brain of a host, comprising applying a therapeutically effective amount of integrin α _v β ₃ and α _v β ₅ antagonists to the host.

For the purposes of the present invention, ECM-dependent cell adhesion includes any ECM-dependent and integrin _αv- mediated cell adhesion. Examples of such adhesion include, but are not limited to, vitronectin-dependent cell adhesion and tenascin-dependent cell adhesion. One finding of the present invention is that human brain tumors can produce vitronectin and tenascin, and these ECMs play an important role in the adhesion and migration of tumor cells by interacting with integrins. For the purposes of the present invention, inhibition is achieved if tumor cell adhesion to the ECM is reduced.

As used herein, the phrase "therapeutically effective amount" refers to a dose of the antagonist sufficient to reduce ECM-mediated tumor cell adhesion. Cell adhesion can be assayed by methods described herein and methods familiar in the art.

According to one embodiment of the present invention, the antagonist can be a polypeptide antagonist of integrin α _v or a mixture of antibodies against α _v β ₃ and α _v β ₅ respectively. For example, the antagonist may be an RGD cyclic pentapeptide antagonist of integrin alpha _v or a cocktail of monoclonal antibodies known as LM-609 and P1-F6.

Another aspect of the present invention provides a method for inhibiting vitronectin-dependent cell migration of brain tumor cells in the brain of a host, comprising applying a therapeutically effective amount of an α _v β ₃ antagonist to the host.

For the purposes of the present invention, inhibition is achieved if tumor cell migration is reduced.

As used herein, the phrase "therapeutically effective amount" refers to a dose of the antagonist sufficient to reduce vitronectin-dependent tumor cell migration. Cell migration can be detected by methods described herein and methods familiar in the art.

According to one embodiment of the present invention, the antagonist can be a polypeptide antagonist of integrin _αv or a monoclonal antibody having _an immune response against _αvβ3 . For example, the antagonist can be an RGD cyclic pentapeptide antagonist of integrin alpha _v or the monoclonal antibody known as LM-609.

The invention also provides a method for inducing tumor cell apoptosis in the host brain. The method comprises administering to the host a therapeutically effective amount of an integrin antagonist.

For the present invention, if the number of brain tumor cell apoptosis observed in the brain increases after the application of the antagonist, it means that the brain tumor cell apoptosis is induced. Therapeutically effective dose refers to the dose of the antagonist sufficient to induce detectable tumor cell apoptosis in the brain of the treated host. Apoptosis of tumor cells in the brain can be detected by methods described herein or methods familiar in the art.

According to one embodiment of the present invention, the integrin may be α _v , α _v β ₃ or α _v β ₅ . Antagonists may be polypeptide antagonists of _αv .

example

Materials and methods

Materials : Active cyclic RGD pentapeptide EMD 121974, cyclic (Arg-Gly-Asp-D-Phe-[N-Me]-Val) and inactive Control peptide cRAD (EMD 135981). Monoclonal antibodies LM609 and P1F6 have been described (13). Brain tumor cell lines DAOY and U87MG were purchased from ATCC Dr. Rockville. The basic culture medium of human brain capillary endothelial cells was provided by Dr. M. Stins from Children's Hospital of Los Angeles, USA (49). Human vitronectin was purchased from Promega, Madison, Wisconsin, USA.

FACS analysis : FACScan cell counter (Becton-Dickinson, SanJose) was used. Conditions were as previously described (43). The primary antibody was LM609 and P1F6 at a ratio of 1:100, and the secondary antibody was FITC-labeled goat anti-mouse IgG at a ratio of 1:250. Apoptosis was determined according to the manufacturer's instructions (Apo Alert Annexin V-FITC Apoptosis Kit, Clontech, Palo Alto, CA, USA).

Adhesion : The test conditions were as described previously (15). Plate wells treated with non-tissue culture medium were incubated overnight at 4°C with vitronectin (1-20μg/ml PBS), washed and blocked with heat-denatured 1% BSA in PBS solution for 30 minutes, and then rinsed with PBS . Control and test cells pre-incubated with test substances (20 μg/ml) in adhesion buffer for 30 minutes at 37°C were added to wells (5×10 ⁴ cells/well) and incubated for 1 hour at 37°C. After gently rinsing the wells with adhesion buffer, the adhered cells were fixed and stained purple. The dye was dissolved in methanol at an OD of 600°A.

Migration : Transwell polycarbonate filter (8 μm pore size) was incubated with vitronectin in PBS solution (1 μg/ml at the top, 10 μg/ml at the bottom) at 37°C for 30 minutes, blocked with 1% heat-denatured BAS for 30 minutes and blocked with PBS rinse. The test cells (5×10 ⁵ /well) were added to the adhesion buffer containing the test indicator substance (20 μg/ml), and incubated with the bottom chamber containing the adhesion buffer at 37°C for 4 hours. The cells in the upper chamber were removed with a cotton swab, the cells at the bottom of the filter were fixed and stained purple, and the number of migrated cells was determined by counting.

Apoptosis : Adhesion conditions were as above, except that, 12-well plates were covered with vitronectin, and 5×10 ⁵ cells/well were added into adhesion buffer. After incubation at 37°C for 30 minutes, the adhesion buffer was removed and a buffer containing 20 μg/ml active cRAD peptide was added and incubated for a further 4 hours. Attached cells were trypsinized, and together with free cells in the supernatant, apoptotic cells were detected using the Apo Alert Annexin V-FITC Apoptosis Kit.

CAM analysis : Egg donors and formulations have been described previously (23). Tumor cells were suspended in 50 μl PBS, the concentration of DAOY cell line was 4×10 ⁶ cells/egg, and the concentration of U87MG cell line was 3.5×10 ⁶ cells/egg. Cells were grown into tumors within 7 days, harvested under sterile conditions, trimmed to a similar size, and seeded onto CAMs of 10-day-old embryos. The active or inactive peptide (100 μg/egg) was then injected intravenously into the CAM. Tumors were photographed in situ after 7 days of growth, harvested, weighed, fixed in 4% buffered formalin, and embedded in paraffin. Serial sections were stained with hematoxylin and eosin.

Brain tumor model : We have previously described the details of the model (31). Intracerebral injection of tumor cells (10 ⁶ /10 μl) was performed at the mentioned coordinates. Intraperitoneal treatment with active cRGD or inactive cRAD peptide (100 μg/50 μl/mouse) was initiated 3 days after implantation of U87MG and 10 days after implantation of DAOY, and repeated daily thereafter until dyscrasia and/or dying state occurred. Then the animals were sacrificed with C0 ₂ anesthesia, the brain tissue was excised and quick-frozen with liquid nitrogen, or fixed with buffered formalin, embedded in paraffin and sectioned for hematoxylin-eosin staining.

Subcutaneous tumor growth : Subcutaneous tumor growth was the subcutaneous injection of tumor cells (10 ⁶ /mouse) under the right shoulder pad of mice immediately after intracerebral injection.

Animal testing : Animal testing was performed in accordance with NIH guidelines and was approved by the local animal protection committee.

test

Angiogenesis on the CAM

To assess the effect of α _v antagonism on brain tumor-associated angiogenesis, chicken chorioallantoic membranes (CAMs) were inoculated with DAOY and U87MG human brain tumor cells. Tumors were allowed to grow for 7 days before they were excised and re-seeded on fresh CAMs. Twenty-four hours after transplantation, active cRGD peptide and control peptide (cRAD) were injected intravenously into the CAM. Tumors were weighed and analyzed for angiogenesis after 6 days of continued growth. By stereomicroscopy, tumors receiving the control peptide showed extensive angiogenesis, whereas tumors treated with the _αv antagonist showed marked inhibition of angiogenesis (Fig. 1a). Tumor growth was also inhibited by _αv antagonists. Control tumors had an 80% increase in weight compared to control tumors, whereas _αv antagonist-treated tumors had a 30% decrease in weight (Fig. 1b). These data suggest that inhibition of tumor growth by _αv antagonists results from disruption of tumor-associated angiogenesis.

Figures 1a and 1b show inhibition of angiogenesis (a) and tumor growth on CAMs by _αv antagonists. Tumors grown on CAMs were harvested and trimmed to the same size and placed on fresh CAMs of 10-day-old eggs. Next, chicks were injected intravenously with 100 μg of active _αv antagonist or control peptide and allowed to grow for 6 days. Eggs receiving the control peptide exhibited significant angiogenesis (a) (DAOY=A, U87MG=C), while significant inhibition of neovascularization was observed in eggs receiving the active peptide (DAOY=B, U87MG=D). Tumors located on the CAM were weighed at the beginning and end of the experiment (b). Both tumors treated with the control peptide increased in weight by 80% (left, n=8), while those treated with the active peptide only increased in weight by 30% (right, n=8).

Orthotopic tumor models

DAOY and U87MG cells (10 ⁶ /mouse) were injected stereotaxically into the right frontal cortex of nu/nu mice in order to establish a system that recapitulates the brain microenvironment. This method allowed for the establishment of a highly reproducible model of human brain tumor generation and the ability to examine tumor growth and mouse survival prior to administration of _αv antagonists (Figures 2a and 2b). Figure 2 shows tumor size (A) and survival of mice (B) after intracerebral injection of DAOY and U87MG cells. U87MG cells grew rapidly and reached a state of about 6 mm in 6 weeks, while DAOY cells grew slowly and reached a diameter of 5.5 mm in 9 weeks (A). All animals died before 9 weeks (B). n=5 or 6 for each time point.

To test the effect of the cRGD peptide in this model, tumors were first grown for 7 days (DAOY) or 3 days (U87MG) and given daily ip injections of _αv antagonists (100 μg/mouse) or inactive control peptides for 3 weeks. At the time of animal sacrifice (4 weeks of total tumor growth), there was very pronounced brain tumor growth in control mice, with an average size of 3 mm (DAOY) and 5.5 mm (U87MG). The mice in the treatment group had no tumor growth or only a very small amount of tumor cells remained on the surface of the ventricle or dura mater under the microscope (Fig. 3a and 3b). Figures 3a and 3b show the histopathological appearance of orthotopically injected brain tumor cells DAOY (a) and U87MG (b) treated daily with inactive (A) or active peptides (BD). Huge intracerebral tumors (arrowheads) were seen in control animals (A), whereas no tumors (B) or only microscopic residual tumors (arrowheads) were present in _αv antagonist-treated animals (CD).

The body weight of the treated mice remained at the baseline level of 21 g, while the body weights of the DAOY and U87MG control mice were 16 g and 15 g, respectively, which was 5-6 g lower than the baseline level (see Table 1). The average time for neurological symptoms in the control group was 2.5 weeks for the mice in the U87MG group and 4.5 weeks for the mice in the DAOY group, while the mice in the treatment group did not show neurological symptoms at any time.

Table 1

cell line Tumor size (mm) Mouse tumor (g) brain Subcutaneous tissue DAOY control peptide 3.0±0.71 18±1.41 16±1.3 active peptide NM 17±0.84 21±0.89 U87MG control peptide 5.5±0.55 20±1.0 15±0.45 active peptide NM 19±0.77 20±0.71

NM = not measurable

Survival tests were also performed on mice. Tests were performed when mice were sacrificed due to their moribund condition. All control animals were sacrificed before 6 weeks of tumor growth, with the majority (>50%) of both control groups sacrificed within 3-4 weeks (Figure 4). Figure 4 shows the mice with orthotopic implanted brain tumors, the U87MG group started from the third day, and the DAOY group started from the seventh day, after receiving active or inactive peptide intraperitoneal injection treatment (100μg/mouse/day) Survival situation. The number of samples (n) in both the DAOY control group and the U87MG control group was 16 mice, the DAOY treatment group was 32 mice, and the U87MG treatment group was 24 mice. Figure 4 shows that none of the animals in the treatment group had to be sacrificed, and the mice in the DAOY treatment group have now survived 24 weeks since the injection of tumor cells, and the mice in the U87MG treatment group have survived 15 weeks. None of the animals in the treatment group developed any neurological symptoms, and all animals continued to grow symptomatically.

Ectopic tumor model

To elucidate the effect of the microenvironment on tumor growth controlled by _αv antagonism, we injected the same human brain tumor cells ( ¹⁰ cells/nude mouse) subcutaneously in mice before treatment initiation, and three days after tumor cell injection (U87MG) and Day 7 (DAOY) treatment with active and control peptides (100 μg/mouse/day, ip). Under these conditions, tumor growth was not inhibited by the active peptide, and after 6 weeks both treated and non-treated groups appeared identical at the macroscopic and microscopic levels, with extensive angiogenesis (data not shown). This shows that the inherent characteristics of the detected brain tumor cells are not enough to make them sensitive to cRGD peptide antagonists, and they must be inhibited in a unique brain microenvironment.

To further investigate this finding, we simultaneously injected DAOY and U87MG cells intracerebrally and subcutaneously prior to the initiation of treatment with the same peptide treatment as described in the orthotopic model. Figure 5 shows the effect of _αv antagonists on orthotopically (brain) and ectopically (subcutaneously) grown DAOY cells. Tumor cells (10 ⁶ ) were injected intracerebral and subcutaneously, and treatment with inactive (a) or active (b) peptides was initiated on the seventh day. Photography was performed at 4 weeks. Subcutaneous tumors were similar in size (A) and degree of vascularization (B) in animals receiving inactive (a) or active (b) peptides. In both cases, subcutaneous tumors grew into the underlying muscle layer (arrows) (C). In contrast, the control group developed giant brain tumors (arrowheads), whereas mice treated with the active peptide had no brain tumors or only microscopic residual tumors (D). Thus, Figure 5 shows that subcutaneous tumors in both the control and treatment groups grew to an average size of 18 mm ³ with marked extensive angiogenesis (Figure 5). Control mice also developed giant brain tumors, similar in size to those previously observed in orthotopic tumor models, with clinical evidence of neurological damage (Table 1). In contrast, mice treated with the active peptide survived without neurological symptoms, with no intracerebral tumor growth or microscopic clusters of tumor cells at autopsy (Fig. 5). However, subcutaneous tumors in this group of mice grew aggressively and necessitated sacrifice at 10 weeks. This demonstrates that tumors growing in the brain respond to cyclic RGD peptide treatment, whereas similar tumors growing in the subcutaneous area do not.

Integrin expression

Vitronectin is a matrix protein that influences a variety of biological responses in the brain and subcutaneous regions, is abundant in tissues outside the nervous system but is rarely produced in glial and neuronal tissues (15,20) . However, glioblastoma cells synthesize vitronectin, which is important for both their attachment and dissemination (15). In other words, vitronectin promotes the adhesion and migration of endothelial cells to the tumor bed, thereby enhancing angiogenesis. Since cell attachment to vitronectin is mediated by integrins α _v β ₃ and α _v β ₅ , we performed FACS analysis to determine the Whether human brain endothelial cells express integrins _αvβ3 and _αvβ5 (Figure _6a ) _.

Figures 6a-6d show the profile of integrins (a), the effect of α _v antagonists on the adhesion of brain tumor and brain capillary endothelial cells to vitronectin (b), the effect of brain tumor and brain capillary endothelial cells on vitronectin Effects on cell migration (c) and viability of brain tumor and brain capillary endothelial cells in vitronectin (d). Both DAOY and U87MG tumor cells and brain capillary endothelial cells express integrins α _v β ₃ and α _v β ₅ (a). Adhesion to vitronectin was significantly inhibited by the active peptide _, also _when the active peptide was combined _with antibodies to _αvβ3 and _αvβ5 , but the control peptide or _antibodies specific to _αvβ3 and _αvβ5 alone There is no obvious effect when used. _Migration of cells in vitronectin gradients was completely abolished by _αvβ3 antibody and _αv antagonist, but not inhibited by control peptide and _αvβ5 antibody _. Cell viability assays were performed by allowing cells to attach to vitronectin for 1 hr, exposing them to control peptide or _αv antagonist (20 μg/ml) (d) for 4 hr, respectively, and mixing attached and floating cells , Apoptosis detection by FACS with FITC-labeled anti-Annexin V and propidium iodide. Apoptosis was rarely observed in cultures exposed to the control peptide (d, left), whereas brain capillary endothelial cells and DAOY cells both showed high numbers of apoptotic cells (d, right).

Figures 6a-6d show that the expression level _{of αvβ3} is higher in U87MG cells than in DAOY cells, but the _expression level of _αvβ5 is higher in the latter. HBEC cells grown in VEGF _- containing medium expressed high levels of _αvβ5 , while in short-term culture, 50% _of the cells expressed _αvβ3 . These cells were also subjected to FACS analysis after 2 days of culture on vitronectin-coated plates. In these cases, expression _{of αvβ3} and _αvβ5 was unchanged (data not shown) _.

vitronectin adhesion

To determine whether α _v β ₃ and α _v β ₅ could regulate the adhesion of DAOY, U87MG and HBEC to vitronectin, cells were plated in vitronectin-coated wells in the presence or absence of α _v β ₃ and α Adhesion was performed in the presence of _{v β5} blocking receptors (LM-609 and P1-F6) or cRGD peptide. P1-F6 minimally inhibited the attachment of DAOY cells, and LM-609 had no effect on the attachment of any cells. When P1-F6 and LM-609 were co-incubated or cRGD peptide alone, the adhesion of all cells was significantly inhibited (Fig. 6). These data suggest that blocking either integrin alone is not sufficient, _and dual blockade _{of αvβ3} and _αvβ5 is necessary to significantly inhibit vitronectin-dependent cell adhesion.

vitronectin migration

To understand whether _αvβ3 and _αvβ5 could regulate adhesion-independent _migration on vitronectin, we tested vitronectin-coated membranes in Boyden chambers in the presence of blocking _antibody or cRGD The same cells were tested above. P1-F6 did not affect migration, and LM-609 and cRGD significantly inhibited the migration of all three types of cells (Fig. 6c). Because LM-609 fails to block adhesion in the absence of P1-F6, this anti-migratory effect must be through other pathways that do not depend on the ability to respond to adhesion. In addition, LM-609 blocked migration as well as cRGD, suggesting that _{αvβ3 -} mediated cell signaling is a critical component in the tested brain tumor and tumor endothelial cell migration assays.

apoptosis

High-grade astrocytomas produce vitronectin at the most anterior invasive margin, suggesting that this protein plays an important role in brain tumor cell attachment, migration, and even survival (15). We have previously shown that pre _- incubation of brain tumor or capillary cells with cRGD peptide or anti- _αvβ3 and _αvβ5 antibodies prevents _cell adhesion to vitronectin. After the same inhibition of integrin-dependent attachment to matrix proteins has been demonstrated, endothelial and melanoma cell apoptosis has also been demonstrated (26-28). To determine whether _αv antagonists could induce apoptosis of these cells after detachment from vitronectin. DAOY cells and primary HBEC cells were seeded in adhesion buffer on vitronectin-coated wells (1 μg/ml) and adhered at 37°C for 30 minutes. The buffer was then exchanged with a buffer containing cRGD or cRAD peptide (control) at a concentration of 20 μg/ml, followed by incubation for 4 hours. Attached cells were mixed with floating cells after trypsinization, and the number of apoptotic cells was determined by FACS using anti-Annexin V-FITC antibody and propidium iodide (Clontech Apo alert Annexin V-FITC Apoptosis Detection Kit). Cells exposed to the control peptide cRAD had little apoptosis, and cRGD-treated DAOY box HBEC cells all had significant apoptosis (Fig. 6d). These data suggest that the cRGD peptide not only detaches cells from vitronectin, but also induces further apoptosis.

Effects of Pentapeptide on the Adhesion of Brain Tumor Cells to Different ECM Substrates

Figure 7 shows the effect of cyclic pentapeptide on the adhesion of tumor cells to ECM proteins. Non-tissue culture dishes were incubated with vitronectin, tenascin, fibronectin or collagen I (10 μg/ml) at 37°C for 1 hour, and then washed with PBS. After washing, inject 5×10 ⁵ cells/well into the culture plate and incubate at 37°C for 16 hours. The cultures were rinsed and added to adhesion buffer containing 20 μg/ml cyclopentapeptide or control peptide and incubated for an additional 2-24 hours. The culture was washed twice with adhesion buffer and stained with crystal violet for OD600 detection. The more adherent cells, the higher the OD value. Data represent adherent cells after 8 hours of incubation. U87 = glioblastoma, DAOY = medulloblastoma.

As illustrated in Figure 7, the adhesion of cells detached from vitronectin and tenascin was mediated by α _v integrin, while that of cells detached from collagen and fibronectin was not, and they were associated with non-α v _v Integrin interactions. Similar data were also obtained in brain capillary cells (not shown).

Effects of cell deattachment on cell survival

Figure 8 shows apoptosis of brain tumor and capillary cells grown on ECM and exposed to pentapeptide. The test conditions are shown in Figure 7, except that adherent cells are lysed and adherent cells are mixed after trypsinization. These cells were washed, suspended in 50 μl PBS, and cytospinned on coverslips. After fixing with 4% paraformaldehyde, cells were stained for apoptosis with Boehringer apoptosis kit. The results represent the proportion of apoptotic cells in the total number of cells. Assays were performed after 24 hours of co-incubation with the peptides.

The effect of cell deattachment on cell survival is shown in Figure 8. As shown, cells exposed to active peptides for 24 hours and grown on vitronectin or tenascin had an increased number of dead cells compared to control cells. In contrast, pentapeptide did not alter the survival of cells grown on collagen I or fibronectin. Reduced cell survival was seen in all tumor cell types and in brain capillary cells. Similar data were obtained when cells were stained for an early cell death marker expressed on the cell surface (Annexin-V, data not shown).

Production of ECM proteins in human brain tumors

As mentioned above, brain tumors produce vitronectin and tenascin in humans, and these substrates are hypothesized to improve tumor cell survival and enhance their invasiveness. We tested the production of these proteins in our brain tumor model.

Figure 9 shows the immunohistochemical appearance of U87 brain tumors transplanted into the forebrain of nude mice and treated with active (anti-α _v antibody) or control peptide. Mice received tumor cell injections (10 ⁶ cells/mouse), and treatment with active or inactive peptides (100 μg/mouse/day) was started on the seventh day after injection. Tumors were resected after 2 weeks of treatment, fixed with buffered formalin, embedded in paraffin, and sectioned at 5 μm. Detection of CD31 (capillary cell marker) expression on sections with rat anti-mouse monoclonal antibody (Phammgen), mouse anti-human vitronectin monoclonal antibody (Sigma), and mouse anti-human tenascin antibody (Neo-Marker) , and the Boehringer apoptosis kit was used for dead cell detection. CD31 = capillary marker, VN = vitronectin, TN = tenascin, APO = apoptosis.

As shown in Figure 9, brain tumors did produce vitronectin and tenascin, and their origin in tumor cells was confirmed using antibodies specific for the human proteins. The application of active pentapeptides has an effect on the synthesis of these proteins. Additionally, we were able to demonstrate that brain tumor cells produced these proteins in tissue culture (data not shown). Thus, our mouse model mimics the conditions of human brain tumors.

The anti-angiogenic effect of pentapeptide is shown at the top of Figure 9, where we stained tissue sections for a capillary cell-specific marker called CD31. Tumors treated with the control peptide had many vessels that stained positive for this marker, whereas such vessels were rarely seen in tumors treated with the active pentapeptide (p<0.05), suggesting capillary growth inhibition. Figure 9 bottom shows staining of apoptotic (dead) cells. The method is described in Figure 8. The tumors treated with the active pentapeptide had significantly more dead cells than the tumors treated with the control peptide (p<0.02). Glioblastoma U87 was photographed and similar data were obtained in medulloblastoma DAOY (not shown).

The Link Between Cell Death and Tumor Cell Apoptosis

To demonstrate that this increased cell death was due to direct tumor cell apoptosis rather than capillary growth inhibition, we seeded mouse brains with melanoma cells expressing (M21 Lα _v positive) or not expressing ( M21 _Lαv negative) _αv integrin. If mice seeded with αv _- positive cells and treated with the active pentapeptide survive longer than mice treated with the control peptide, direct tumor cell apoptosis should be responsible.

Table 2 shows the survival of mice bearing _αvβ3 negative and positive tumors treated with RGDfV _. Tumor cells (10 ⁶ ) were injected into the forebrain of 6-week-old nude mice, and treatment with active or inactive peptides (100 μg/mouse/day) was initiated on the third day. Mice were sacrificed when dyscrasias developed, and the presence of tumors was demonstrated by autopsy.

Table 2

cell line Survival period (days) RGDfV (active) RADfV (control) M21Lα _v negative 15.7±3.8 15.3±3.3 M21Lα _v positive 36.5±2.9 17.3±1.9

As shown in Table 2, mice seeded with αv _- negative melanoma cells survived for 15 days, independent of the treatment received. In contrast, the survival of mice bearing αv _- positive tumors increased to 36 days when treated with the active pentapeptide, while mice treated with the control peptide survived 17 days. This suggests that direct tumor cell apoptosis should be the cause of tumor cell death.

Taken together, the data discussed above support the hypothesis that cyclic pentapeptides induce direct tumor cell death.

discuss

Brain tumors are highly angiogenic, and their continued growth depends on the generation of new blood vessels. Therefore, anti-angiogenesis may be an important therapeutic strategy against these malignancies. Since _expression of integrins _αvβ3 and _αvβ5 on the vascular endothelium _is activated during angiogenesis, they are candidate anti-angiogenic targets (12, 13). Their role in angiogenesis has been demonstrated by previous studies showing that angiogenesis induced by bFGF and TNF-α in the CAM can be blocked by the α _v β ₃ antibody LM-609 or α _v β ₃ and α Inhibited by the antagonist cyclic pentapeptide of _v β ₅ (21, 23). In either case, the anti-angiogenic effect is thought to be achieved by preventing the interactions necessary for _αv -matrix protein binding, thereby inducing apoptosis in endothelial cells (28-30). In this study, we employed an antagonist of α _v β ₃ and α _v β ₅ similar to a specific RGD cyclic peptide (EMD121974) to demonstrate its inhibitory effect on angiogenesis induced by two human brain tumor cell lines DAOY and U87MG in CAM. Inhibition. cRGD treatment not only inhibited angiogenesis in CAMs, but further resulted in tumor necrosis and tumor shrinkage.

To test whether a similar response could be obtained in a model that mirrors the brain microenvironment, brain tumor cells were injected stereotaxically into the forebrain of nude mice. U87MG glioma and DAOY medulloblastoma cells were chosen for the study due to their reactivity to the peptide in the CAM assay and their extensive invasiveness and angiogenesis demonstrated by previous in vivo assays (31, 32). In this orthotopic model, cRGD again significantly inhibited the growth of U87MG and DAOY brain tumors. Control mice succumbed to tumor progression and were found to have highly aggressive tumors with a mean volume greater than ³ mm. Mice treated with cRGD survived without evidence of disease, with no viable tumors detectable except for residual cells along the implantation site or small clusters of cells on the ventricle and dural surface. All residual tumors clustered in the treatment group were less than 1 mm ³ and thus did not achieve a host angiogenic response. Alternative angiogenesis inhibitors, such as thrombospondin-1, TNP-470, and platelet factor 4, have also been shown to inhibit the growth of experimental brain tumors, but most of these inhibitions are limited in subcutaneous xenografts. limitations, or require direct drug delivery to the tumor bed by stereotaxic injection (32-34). Peptide antagonists of _αvβ3 were shown to inhibit tumor growth by 80% following intraperitoneal application in a SCID mouse/rat _Leydig cell subcutaneous tumor model (35). Angiostatin, an anti-angiogenic agent through an unknown mechanism of action, has also been shown to be quite potent against intracerebral C6 and 9L rat glioma xenografts (36). In contrast to our study, treatment with angiostatin (1 mg/mouse/day) or peptide analog antagonist (2 mg/mouse/day) was started immediately after tumor cell seeding. Our study shows nearly total elimination of viable tumor grafts and is the first to show inhibition of brain tumor growth and angiogenesis through an integrin-antagonistic mechanism.

Interestingly, in our study, both DAOY and U87MG tumors grown subcutaneously showed no or very low response to cRGD treatment. This phenomenon underscores the importance of the extracellular environment in regulating host-tumor-cell integrin responses. Angiostatins were shown to be equally inhibitory against sc and ic injected brain tumor cells, suggesting that this compound inhibits angiogenesis through a mechanism distinct from that of the RGD peptide (36). One possible explanation for the difference in the inhibitory effect of cRGD on tumor growth is that subcutaneous endothelial cells may be fundamentally different from endothelial cells in the CNS microvasculature, and angiogenesis in the former is less sensitive to integrin antagonism mechanisms. This hypothesis is supported by recent findings that in mice with high expression of _αv , only the capillary cells of the brain and intestine were abnormal, while the rest of the circulatory system was normal (37). In other words, matrix proteins that are ligands for endothelial cell integrins are preferentially expressed by tumor cells, depending on whether they are orthotopic or ectopic tumors. For example, human glioblastoma cells grown subcutaneously were not found to produce vitronectin, but exposure to the brain microenvironment induced expression of the vitronectin gene (15). Normal brain tissue appears to alter the expression of ECM proteins when invaded by glioma cells (38). Unlike extraneural sites, vitronectin is normally present in brain tissue, and small changes in this protein in the CNS can profoundly affect cellular responses. Studies have shown that the motility of endothelial cells is dependent on the concentration of vitronectin and the distance between the cell-matrix contact points that allow cell motility (39, 40).

In our study, the anti-tumor effects of the peptides were generally higher than those of other anti-angiogenic agents. Since many tumors also express _αvβ3 and _αvβ5 integrins (including U87MG and DAOY), _{the effects} of integrin antagonists would not be restricted to the host endothelium, although the importance of specific integrins in tumor cell responses is unclear (41). Previous studies have shown that attachment of glioma, breast cancer, melanoma and HT29-D4 colon adenocarcinoma cells to vitronectin is dependent on _αv integrin (42-45). Vitronectin is produced by tumor and basal cells and is abundant at sites of tumor invasion and neovascularization, including malignant brain tumors (46, 47). Therefore, besides supporting endothelial cell _survival , vitronectin is also critical for enhancing the adhesion and invasion of tumor cells expressing _αvβ3 and _{αvβ5 .} In the SCID mouse/human breast cancer chimeric model, tumor invasion was significantly reduced after application of anti-α _v β ₃ antibody LM-609, indicating that α _v β ₃ blockers have a direct inhibitory effect on tumors independent of angiogenesis.

To address this question, we examined cell adhesion and migration on vitronectin in the presence of _αv antagonists using DAOY, U87MG and isolated primary human brain endothelial cells (HBEC) (49). Anti-α _v β ₃ antibody LM-609 inhibited the migration of U87MG, DAOY and HBEC on vitronectin, combined with anti-α _v β ₅ antibody P1-F6 inhibited the adhesion of these cells to vitronectin. cRGD alone significantly inhibited the adhesion and migration of all three cell types to vitronectin. In a similar study, melanoma cell invasion was enhanced by vitronectin and blocked by RGD peptide (50). Finally, cRGD treatment not only resulted in increased apoptosis in HBEC cells, but also in DAOY and U87MG tumor cells. These results suggest that, in addition to its anti-angiogenic function, cRGD may directly inhibit tumor defects and proliferation.

Our data further show that cyclic pentapeptide can inhibit the adhesion of brain tumor cells to different ECM substrates such as vitronectin or tenascin. This inhibition of adhesion can lead to cell death (apoptosis) in brain tumor cells and brain capillary cells. This effect was restricted to ECM vitronectin and tenascin. We also demonstrate that the increase in cell death is due to direct apoptosis rather than as a result of inhibition of capillary growth. In other words, one of the findings of the present invention is that cyclic pentapeptides induce direct tumor cell death.

In conclusion, this study demonstrates that targeted antagonism against integrins, especially α _v β ₃ and α _v β ₅ , can substantially inhibit brain tumorigenesis in vivo, thus providing an important new approach for the treatment of brain tumors. Our results also suggest that the microenvironment is critical to tumor behavior and determines its responsiveness to such direct biological treatments. Finally, we show that integrin antagonism can exert an anti-angiogenesis-independent anti-tumor effect that enhances the inhibition of tumor growth.

The foregoing content is intended to illustrate rather than limit the scope of the present invention. In fact, other embodiments can be conceived and suggested by one skilled in the art in light of the teachings herein without undue experimentation.

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Claims (5)

1. integrin antagonists is used for suppressing the purposes of the medicine of host's brain tumor growth in preparation, and wherein said integrin antagonists is selected from: anti-α _vβ ₃Antibody, anti-α _vβ ₅Antibody and anti-respectively α _vβ ₃And α _vβ ₅The combination of antibody.

2. the purposes of claim 1, wherein said antagonist is α _vβ ₃The immunologic opsonin monoclonal antibody.

3. the purposes of claim 2, wherein said monoclonal antibody has the immunoreation feature of the monoclonal antibody of LM-609 by name.

4. the purposes of claim 1, wherein said anti-respectively α _vβ ₃And α _vβ ₅Antibody be combined as α _vβ ₃Immunologic opsonin monoclonal antibody and α _vβ ₅The combination of immunologic opsonin monoclonal antibody.

5. the purposes of claim 4, wherein said α _vβ ₃The immunologic opsonin monoclonal antibody have the immunoreation feature of LM-609 monoclonal antibody by name and described α _vThe immunologic opsonin monoclonal antibody of β 5 has the immunoreation feature of P1-F6 monoclonal antibody by name.

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